A microfluidic chip for detecting nucleic acid of microorganisms and an automatic detection box and an application method thereof

Abstract

The microfluidic chip for detecting nucleic acid of microorganisms of the present invention has a first solenoid valve that controls the on-off of the inlet and outlet of the eluent, and the eluent flows out from the outlet through the sample chamber of the sample injection chip, and enters the degassing channel through the sample inlet; the second solenoid valve controls the on-off of the outlet of the degassing channel and the inlet of the lysis pool, and the sample completes the release of nucleic acid in the lysis pool; the third solenoid valve controls the on-off of the outlet of the lysis pool and the inlet of the reaction detection pool, and the reaction detection pool is provided with a plurality of reaction chambers, and the freeze-dried powder of the LAMP-CRISPR reaction system is pre-set in the reaction chamber, and the constant temperature amplification of nucleic acid is completed in the reaction chamber. The chip card box of the present invention is fully integrated, and has low requirements for operators; a swab is used for direct sample loading, and no manual extraction process is required; the chip card box is plug-in and unplug design, and is convenient for installation and replacement.

Description

Technical Field

[0001] The present invention belongs to the field of microbial detection, and specifically relates to a microfluidic chip and a detection kit for microbial nucleic acid detection and an application method thereof. Background Art

[0002] The current microbial testing process includes sample collection, storage and transportation, as well as laboratory testing steps. Laboratory testing is specifically divided into multiple links such as sample pretreatment, dilution, cultivation and testing. The operation process is complex and delicate, involving techniques such as sample pretreatment, nucleic acid amplification and detection. This series of operations places strict requirements on the operating environment. Not only does it require professional operators to ensure the accurate execution of each step, but it also requires specific equipment and environmental conditions. These limiting factors have, to a certain extent, hindered the widespread application of microbial testing in different scenarios.

[0003] This category has the following deficiencies: Microbial testing is limited by the operating environment and professionals, and there are many deficiencies. Specifically, the equipment and technology levels are backward, which makes it difficult to meet the needs of efficient and accurate testing; the lack of new testing methods makes it difficult to effectively detect some microorganisms; the quality of testing personnel is uneven, and some personnel lack professional theoretical knowledge and lack a strong sense of responsibility, which affects the accuracy of the test results; the quality of the specimens does not meet the standards, which may lead to deviations in the test results; the test plan is unreasonable and fails to fully consider the characteristics and testing needs of different microorganisms; and the lack of communication between the laboratory and the clinic affects the application of the test results and the treatment of patients. These problems have jointly restricted the development and application of microbial testing.

[0004] Therefore, there is an urgent need to automate, miniaturize and facilitate testing so that it can be carried out on-site in different scenarios. Summary of the invention

[0005] In order to overcome the problems in the prior art, the present invention provides a microfluidic chip and an automatic detection box for detecting microbial nucleic acid, which use a microfluidic chip as a carrier and have a fully closed and automated experimental process.

[0006] The present invention provides the following technical solutions:

[0007] A microfluidic chip for detecting nucleic acid of microorganisms comprises a degassing channel, a lysis pool and a reaction detection pool integrated on the microfluidic chip, the microfluidic chip further comprising a first solenoid valve, a second solenoid valve and a third solenoid valve, the first solenoid valve controls the connection and disconnection of an eluent inlet and an outlet, the eluent flows out from the outlet through a sample chamber of a sampling chip, and enters the degassing channel through a sample inlet; the second solenoid valve controls the connection and disconnection of the degassing channel outlet and the lysis pool inlet, a heating component is connected above the lysis pool, and nucleic acid release of the sample is completed in the lysis pool; the third solenoid valve controls the connection and disconnection of the lysis pool outlet and the reaction detection pool inlet, the reaction detection pool is provided with a plurality of reaction chambers, freeze-dried powder of a LAMP-CRISPR reaction system is pre-set in the reaction chamber, a heating component is connected above the reaction chamber, and constant temperature amplification of nucleic acid is completed in the reaction chamber.

[0008] Furthermore, the sample chamber is placed in the injection chip, and the sample chamber is connected to the injection chip by a sample loading port, the sample loading port is switched by a sealing screw, the sample chamber inlet is connected to a liquid inlet pipe, the liquid inlet pipe is connected to an eluent outlet of the microbial nucleic acid detection microfluidic chip, the sample chamber outlet is connected to a liquid outlet pipe, and the liquid outlet pipe is connected to a sample inlet set on the microbial nucleic acid detection microfluidic chip.

[0009] Furthermore, a liquid storage bag for the eluent is provided on the microbial nucleic acid detection microfluidic chip, and the liquid storage bag for the eluent is connected to the eluent inlet via a peristaltic pump.

[0010] Furthermore, a water-proof and breathable pad is provided on the degassing flow channel, and a pressure plate is provided on the water-proof and breathable pad, and the degassing flow channel is sealed by the pressure plate, and the width of the degassing flow channel is 1 to 3 mm.

[0011] Furthermore, the lysis pool is a spindle-shaped pool body, and the volume of the lysis pool is larger than the total volume of each of the reaction chambers.

[0012] Furthermore, the inlet of the reaction detection pool is connected to the reaction chamber through a plurality of liquid inlet branches, the volumes of the reaction chambers are the same, the reaction chambers are connected to the liquid outlet branches, the outlets of the liquid outlet branches are connected to the gas outlet, a water-proof and breathable pad is provided on the gas outlet, the gas outlet is sealed by a gas outlet pressure plate, and a light-transmitting window is provided at the bottom of the reaction chamber.

[0013] Furthermore, the inlet of the reaction detection pool is connected to each of the liquid inlet branches through a liquid inlet main pipe, and a bypass liquid flow channel is also connected in parallel on one side of the liquid inlet branch. The diameter of the bypass liquid flow channel is wider than the diameter of the liquid inlet branch, the outlet of the bypass liquid flow channel is connected to the air outlet, and the volumes of the liquid outlet branches are the same.

[0014] Furthermore, the heating component is provided with a heat conducting plate, a heating plate and a heat insulating plate from bottom to top, a temperature sensor is provided in the heat conducting plate, and is sealed and fixed at a corresponding position on the microfluidic chip by a sealing ring.

[0015] Furthermore, an elliptical liquid level detection pool is provided in the liquid inlet pipe of the lysis pool, and metal electrodes are provided at both ends of the liquid level detection pool, one end of the electrode is in the detection pool, and the other end leaks out of the chip and is connected to a wire.

[0016] Furthermore, the liquid inlet pipeline of the lysis tank is provided with a serpentine pipeline.

[0017] Furthermore, the connecting pipeline between the sample inlet and the degassing flow channel passes through the water-proof and air-permeable pad, and the pipeline passing through the water-proof and air-permeable pad is sealed.

[0018] An automatic detection box including a microfluidic chip for detecting microbial nucleic acid, wherein the automatic detection box is detachably connected to the top of a detection device box body by a chip box body, the microbial nucleic acid detection microfluidic chip is fixed inside the chip box body, the sampling chip is fixed inside the chip box body, the sampling port abuts against the surface of the chip box body, and a sealing screw protrudes outside the chip box body; a fluorescence detector is arranged in the detection device box body, and the fluorescence probe of the fluorescence detector corresponds to the position of the reaction chamber. A main control board is also arranged in the detection device box body, and the main control board is used to electrically control various electrical components.

[0019] Furthermore, the fluorescence detector is connected to the detection device box through a connecting rod, and the connecting rod is provided with a rotating wheel and a belt. The rotating wheel is driven by a stepper motor to drive the belt to rotate, thereby completing the lateral movement of the fluorescence detector under each reaction chamber. The lateral movement range is limited by a photoelectric switch. The fluorescence detector is provided with FAM, VIC, ROX, and CY5 channels.

[0020] Furthermore, the main control board controls the heating of the heating plate and collects the temperature of the temperature sensor in the heat conduction plate, controls the opening and closing of the peristaltic pump and the solenoid valve, reads the liquid detection signal of the liquid level sensor, controls the rotation of the stepper motor and the fluorescence signal collection of the fluorescence detector, and reads the position signal of the photoelectric switch.

[0021] A method for detecting Staphylococcus aureus nucleic acid using an automatic detection kit comprises the following steps:

[0022] Step 1) pre-place LAMP amplification primers of the nuc gene of Staphylococcus aureus, sgRNA targeting the nuc gene of Staphylococcus aureus, AapCas12b protease, fluorescent probe, Bst3.0 polymerase, dNTPs reagent and lyophilized powder of reaction buffer in the reaction chamber; one of the reaction chambers is set as a blank control;

[0023] Step 2) Open the sealing screw, and the swab carrying the sample to be tested enters the sample chamber from the sample loading port, break the swab rod and leave the swab head in the sample chamber, tighten the sealing screw, and the main control board controls the peristaltic pump to pump the eluent in the liquid storage bag into the eluent inlet, open the first solenoid valve, and the eluent flows into the sample chamber of the sample injection chip for elution, and then enters the degassing flow channel from the sample inlet to degas the eluent;

[0024] Step 3) The main control board controls the peristaltic pump to continue pumping liquid. After the electrode detects that the eluent flows into the liquid level detection pool, the timing is started, and the peristaltic pump is controlled by time to transport the eluent to fill the lysis pool. Then the peristaltic pump, the first solenoid valve and the second solenoid valve are closed, and the heating component connected above the lysis pool is controlled to heat to 90°C, and the lysis is performed for 5-15 minutes to complete the nucleic acid extraction;

[0025] Step 4) The main control board controls the peristaltic pump and the third solenoid valve to open, deliver the lysate into the reaction chamber, then closes the third solenoid valve, and controls the heating component connected above the reaction chamber to heat to 65° C., reacts for 60 minutes, and amplifies the nucleic acid at a constant temperature for 60 cycles, each cycle lasting 1 minute;

[0026] Step 5) The main control board controls the stepper motor to drive the fluorescence detector to pass through the bottom of each reaction chamber in turn, and the fluorescence probe opens the FAM channel to perform fixed-point fluorescence detection. The main control board controls the fluorescence probe to perform a fluorescence signal detection once in each cycle.

[0027] By adopting the above technical solution, the present invention has the following beneficial effects:

[0028] 1. The chip card box of the present invention is fully integrated, with low requirements on operators; the swab is used for direct sample loading, without the need for manual extraction process; the chip card box has a plug-in design, which is convenient for installation and replacement;

[0029] 2. The detection of the present invention adopts one-step thermal lysis, and the lysis time and effect are controllable; multiple detection wells can be combined with different detection items; using LAMP-CRISPR detection technology, the detection sensitivity is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the structure of the microfluidic chip and the sample injection chip for detecting microbial nucleic acid of the present invention;

[0031] Figure 2 This is an exploded view of the structure of the microfluidic chip for detecting microbial nucleic acids of the present invention;

[0032] Figure 3 It is a structural schematic diagram of the sample injection chip of the present invention;

[0033] Figure 4 This is a schematic diagram of the liquid channel structure of the microfluidic chip for detecting nucleic acid of microorganisms of the present invention;

[0034] Figure 5 This is another schematic diagram of the liquid channel structure of the microfluidic chip for detecting microbial nucleic acids of the present invention;

[0035] Figure 6 It is a schematic diagram of the connection of the microfluidic chip for detecting nucleic acid of microorganisms in the automatic detection box of the present invention;

[0036] Figure 7 It is a structural cross-sectional view of the internal components of the automatic detection box of the present invention;

[0037] Figure 8 It is a perspective view of the automatic detection box of the present invention.

[0038] Fig. 9 This is a diagram of the fluorescence detection results in Example 3 of the present invention.

[0039] Wherein: 1. Microbial nucleic acid detection microfluidic chip, 101. degassing channel, 102. lysis pool, 103. reaction chamber, 104. first solenoid valve, 105. second solenoid valve, 106. third solenoid valve, 107. sample inlet, 108. degassing channel outlet, 109. lysis pool inlet, 1010. lysis pool outlet, 1011. reaction detection pool inlet, 1012. eluent outlet, 1013. eluent inlet, 1014. liquid storage bag, 1015. peristaltic pump, 1016. air outlet, 1017. heat conducting plate, 1018. heating plate, 1019. heat insulation plate, 1020. sealing ring, 1021. bypass liquid channel, 1022. liquid level detection pool;

[0040] 2. Sample injection chip, 201. Sample chamber, 202. Sample injection port, 203. Sealing screw, 204. Sample chamber inlet, 205. Sample chamber outlet; 3. Chip box, 4. Detection equipment box, 401. Fluorescence detector, 402. Main control board, 403. Stepper motor, 404. Photoelectric switch DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] Example 1

[0043] like Figure 1-4As shown, the present invention provides a microfluidic chip 1 for detecting nucleic acid of a microorganism, comprising a degassing channel 101, a lysis pool 102 and a reaction detection pool integrated on the microfluidic chip, the microfluidic chip also comprising a first solenoid valve 104, a second solenoid valve 105 and a third solenoid valve 106, the first solenoid valve controls the on-off of the inlet and outlet of the eluent, the eluent flows out from the outlet through the sample chamber 201 of the injection chip 2, and enters the degassing channel through the sample inlet 107; the second solenoid valve controls the on-off of the outlet 108 of the degassing channel and the inlet 109 of the lysis pool, a heating component is connected above the lysis pool, and the sample completes nucleic acid release in the lysis pool; the third solenoid valve controls the on-off of the outlet 1010 of the lysis pool and the inlet 1011 of the reaction detection pool, the reaction detection pool is provided with a plurality of reaction chambers 103, the reaction chamber is pre-set with freeze-dried powder of the LAMP-CRISPR reaction system, a heating component is connected above the reaction chamber, and nucleic acid isothermal amplification is completed in the reaction chamber.

[0044] The sample chamber is placed in the sample injection chip, and the sample chamber is connected to the sample chamber by the sample injection port 202 on the sample injection chip, and the sample injection port is opened and closed by the sealing screw 203. The sample chamber inlet 204 is connected to the liquid inlet pipe, and the liquid inlet pipe is connected to the eluent outlet 1012 set on the microbial nucleic acid detection microfluidic chip. The sample chamber outlet 205 is connected to the liquid outlet pipe, and the liquid outlet pipe is connected to the sample inlet 107 set on the microbial nucleic acid detection microfluidic chip. An eluent storage bag 1014 is provided on the microbial nucleic acid detection microfluidic chip, and the eluent storage bag is connected to the eluent inlet 1013 through a peristaltic pump 1015.

[0045] The peristaltic pump controls the eluent in the liquid storage bag to be pumped into the eluent inlet 1013, the first solenoid valve controls the eluent inlet to be connected to the eluent outlet 1012, the eluent enters the sample chamber, and the eluent flowing out from the outlet 205 of the sample chamber reaches the sample inlet 107, and enters the degassing channel to degas the eluent. The second solenoid valve controls the degassing channel outlet 108 to be connected to the lysis pool inlet 109, the eluent enters the lysis pool, and nucleic acid lysis is performed in the lysis pool. Preferably, the lysis pool is a spindle-shaped pool body, and the volume of the lysis pool is greater than the total volume of each of the reaction chambers. The third solenoid valve controls the lysis pool outlet 1010 to be connected to the reaction detection pool inlet 1011, the lysate enters each reaction chamber 103, and nucleic acid isothermal amplification is performed in the reaction chamber.

[0046] A water-proof and breathable pad is provided on the degassing channel, and a pressure plate is provided on the water-proof and breathable pad. The degassing channel is sealed by the pressure plate. The width of the degassing channel is 1 to 3 mm. The appropriate channel width can ensure the efficiency of bubble removal and ensure that the liquid will not be retained in the channel.

[0047] The inlet 1011 of the reaction detection pool is connected to the reaction chamber through a plurality of liquid inlet branches. The volumes of the reaction chambers are the same. The reaction chamber is connected to the liquid outlet branch. The outlet of the liquid outlet branch is connected to the gas outlet 1016. A water-proof and breathable pad is provided on the gas outlet. The gas outlet is sealed by pressing the gas outlet pressure plate. A light-transmitting window is provided below the reaction chamber. The function of the water-proof and breathable pad is to seal the liquid and allow the gas to pass through. In the process of the lysate filling the reaction chamber, the gas in the chamber can be discharged through the water-proof and breathable pad, and the liquid will be blocked in the liquid outlet branch.

[0048] The heating assembly is provided with a heat conducting plate 1017, a heating plate 1018 and a heat insulating plate 1019 from bottom to top. A temperature sensor is provided inside the heat conducting plate and is sealed and fixed at a corresponding position on the microfluidic chip by a sealing ring 10120.

[0049] Example 2

[0050] like Figure 5 As shown, the entrance of the reaction detection pool is connected to each liquid inlet branch through the liquid inlet main pipe, and a bypass liquid flow channel 1021 is also connected in parallel on one side of the liquid inlet branch. The diameter of the bypass liquid flow channel is wider than the diameter of the liquid inlet branch, and the bypass liquid flow channel outlet is connected to the air outlet, and the volume of the liquid outlet branch is the same. The liquid first flows into the bypass liquid flow channel from the liquid inlet main pipe, and then flows into the liquid inlet branch after filling the bypass liquid flow channel to be distributed into each reaction detection pool. This design can prevent the eluent that exceeds the lysis pool from entering the reaction detection pool, and avoid the uncracked sample from occupying the space of the reaction detection pool, resulting in a decrease in detection sensitivity or even detection failure.

[0051] An elliptical liquid level detection pool 1022 is provided in the liquid inlet pipe of the lysis pool, and metal electrodes are provided at both ends of the liquid level detection pool, one end of the electrode is in the detection pool, and the other end leaks out of the chip and is connected to the wire. The part where the metal electrode contacts the chip remains sealed. This design can ensure that there is enough liquid in the liquid level detection pool to contact the two electrodes, and at the same time, the wider detection pool can reduce the resistance value between the electrodes to ensure the sensitivity of the detection.

[0052] Preferably, the liquid inlet pipeline of the lysis tank can be provided with a serpentine pipeline, which can increase the mixing effect of the liquid during the flow process.

[0053] The sample inlet 107 can also be designed next to the gas outlet, so that the connecting pipeline between the sample inlet 107 and the degassing channel passes through a water-proof and breathable pad, and the water-proof and breathable pad seals the passing pipeline. This design allows the sample degassing and the reaction detection pool to share a water-proof and breathable pad, reducing the difficulty of chip processing.

[0054] Example 3

[0055] like Figure 6-8As shown, the present invention provides an automatic detection box including a microfluidic chip for detecting microbial nucleic acid, and the automatic detection box is detachably connected to the top of the detection device box body 4 by a chip box body 3. The microfluidic chip 1 for detecting microbial nucleic acid is fixed inside the chip box body, the sample injection chip 2 is fixed inside the chip box body, the sample injection port abuts against the surface of the chip box body, the sealing screw protrudes outside the chip box body, and the sealing screw is opened for sample injection.

[0056] The fluorescence detector 401 is arranged in the detection device box, and the fluorescence probe of the fluorescence detector corresponds to the position of the reaction chamber. The detection device box is also provided with a main control board 402, which is used to electrically control various electrical components.

[0057] The fluorescence detector is connected to the detection device box through a connecting rod, and a rotating wheel and a belt are provided on the connecting rod. The rotating wheel is driven by a stepper motor 403 to drive the belt to rotate, thereby completing the lateral movement of the fluorescence detector under each reaction chamber. The lateral movement range is limited by a photoelectric switch 404. The fluorescence detector is provided with FAM, VIC, ROX, and CY5 channels.

[0058] The main control board controls the heating of the heating plate and collects the temperature of the temperature sensor in the heat conduction plate, controls the opening and closing of the peristaltic pump and the solenoid valve, reads the liquid detection signal of the liquid level sensor, controls the rotation of the stepper motor and the fluorescence signal collection of the fluorescence detector, and reads the position signal of the photoelectric switch.

[0059] Example 4

[0060] The present invention provides a method for detecting Staphylococcus aureus nucleic acid using an automatic detection kit, comprising the following steps:

[0061] Step 1) pre-place LAMP amplification primers of the nuc gene of Staphylococcus aureus, sgRNA targeting the nuc gene of Staphylococcus aureus, AapCas12b protease, fluorescent probe, Bst3.0 polymerase, dNTPs reagent and lyophilized powder of reaction buffer in the reaction chamber; one of the reaction chambers is set as a blank control.

[0062] The nucleotide sequences of LAMP amplification primers F3, B3, FIP, BIP, LF, and LB and the nucleotide sequence of sgRNA are shown in Table 1 Based on the LAMP-CRISPR / Cas12b method, combined with microfluidic chips and devices, one-step automated detection of microorganisms can be achieved, which has the advantages of high sensitivity and short detection time.

[0063] Table 1

[0064]

[0065]

[0066] Step 2) Open the sealing screw, and the swab carrying the sample to be tested enters the sample chamber from the sample loading port, break the swab rod and leave the swab head in the sample chamber, tighten the sealing screw, and the main control board controls the peristaltic pump to pump the eluent in the liquid storage bag into the eluent inlet, open the first solenoid valve, and the eluent flows into the sample chamber of the sample injection chip for elution, and then enters the degassing flow channel from the sample inlet to degas the eluent;

[0067] Step 3) The main control board controls the peristaltic pump to continue pumping liquid. After the electrode detects that the eluent flows into the liquid level detection pool, the timing is started. The peristaltic pump is controlled by time to transport the eluent to fill the lysis pool. Then the peristaltic pump, the first solenoid valve and the second solenoid valve are closed, and the heating component connected above the lysis pool is controlled to heat to 90°C and lyse for 5-15 minutes to complete the nucleic acid extraction.

[0068] Step 4) The main control board controls the peristaltic pump and the third solenoid valve to open, deliver the lysate into the reaction chamber, then closes the third solenoid valve, and controls the heating component connected above the reaction chamber to heat to 65°C, reacts for 60 minutes, and amplifies the nucleic acid at a constant temperature for 60 cycles, each cycle lasting 1 minute.

[0069] LAMP reaction: The reaction system is 25 μL, including 2 μL template solution, 8 U Bst 3.0 polymerase, 6 mM MgSO4, 1.4 mM dNTPs, 1x reaction buffer, 0.2 μM F3, 0.2 μM B3, 0.4 μM LF, 0.4 μM LB, 1.6 μM FIP, 1.6 μM BIP. The nucleic acid amplification process is 40 cycles of constant temperature amplification at 65 °C, with 1 minute per cycle.

[0070] LAMP-CRISPR / Cas12b reaction system (20 μL): contains 5 μL LAMP reaction system, 250 nM fluorescent probe ( / 5'-FAM-XXXX-BHQ1-3' / ), 250 mM AapCas12b and 250 mM sgRNA65°C, constant temperature amplification for 60 cycles, each cycle for 1 minute.

[0071] The nuc gene of Staphylococcus aureus was selected as the target region through specific sequence analysis and comparison, and LAMP primers were designed. The complementary sequence of the F2-B2 amplification region of the LAMP primers was used to design the recognition RNA (sgRNA) sequence of AapCas12b. LAMP amplification produces a large number of DNA amplification products containing the nuc gene, which can be used as target DNA for CRISPR / Cas12b reaction, stimulate the trans-cleavage activity of Cas12b, cut the fluorescent probe labeled with single-stranded fluorescent molecules-quenching molecules, and generate fluorescent signals.

[0072] Step 5) The main control board controls the stepper motor to drive the fluorescence detector to pass through the bottom of each reaction chamber in turn, and the fluorescence probe opens the FAM channel to perform fixed-point fluorescence detection. The main control board controls the fluorescence probe to perform a fluorescence signal detection once in each cycle.

[0073] The feasibility of LAMP-CRISPR / Cas12b system detection was evaluated. The results are as follows Fig. 9 As shown, the negative control group was added with DEPC water, and the experimental group was added with a target solution containing the nuc gene. The fluorescence signal intensity of each group was compared after 20 minutes of reaction. The experimental group was significantly higher than the negative control group, proving that the automatic detection kit of the present invention can quickly and accurately identify the nuc gene of Staphylococcus aureus for microbial nucleic acid detection.

[0074] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A microfluidic chip for detecting nucleic acid of microorganisms, comprising a degassing channel, a lysis pool and a reaction detection pool integrated on the microfluidic chip, characterized in that: The microfluidic chip also includes a first solenoid valve, a second solenoid valve and a third solenoid valve. The first solenoid valve controls the connection between the eluent inlet and the outlet. The eluent flows out from the outlet through the sample chamber of the injection chip and enters the degassing channel through the sample inlet; the second solenoid valve controls the connection between the degassing channel outlet and the lysis pool inlet. A heating component is connected above the lysis pool, and the sample completes nucleic acid release in the lysis pool; the third solenoid valve controls the connection between the lysis pool outlet and the reaction detection pool inlet. The reaction detection pool is provided with a plurality of reaction chambers. Lyophilized powder of the LAMP-CRISPR reaction system is pre-installed in the reaction chamber. A heating component is connected above the reaction chamber, and constant temperature amplification of nucleic acids is completed in the reaction chamber.

2. The microbial nucleic acid detection microfluidic chip according to claim 1, characterized in that: The sample chamber is placed in the sample injection chip, and the sample chamber is connected to the sample chamber by a sample injection port on the sample injection chip. The sample injection port is switched by a sealing screw. The sample chamber inlet is connected to a liquid inlet pipe, which is connected to an eluent outlet of the microbial nucleic acid detection microfluidic chip. The sample chamber outlet is connected to a liquid outlet pipe, which is connected to a sample inlet on the microbial nucleic acid detection microfluidic chip.

3. The microbial nucleic acid detection microfluidic chip according to claim 2, characterized in that: A liquid storage bag for the eluent is arranged on the microbial nucleic acid detection microfluidic chip, and the liquid storage bag for the eluent is connected to the eluent inlet through a peristaltic pump.

4. The microbial nucleic acid detection microfluidic chip according to claim 3, characterized in that: A water-proof and air-permeable pad is arranged on the degassing channel, and a pressing plate is arranged on the water-proof and air-permeable pad. The degassing channel is sealed by the pressing plate. The width of the degassing channel is 1 to 3 mm.

5. The microbial nucleic acid detection microfluidic chip according to claim 3, characterized in that: The lysis pool is a spindle-shaped pool body, and the volume of the lysis pool is larger than the total volume of each of the reaction chambers.

6. The microbial nucleic acid detection microfluidic chip according to claim 5, characterized in that: The inlet of the reaction detection pool is connected to the reaction chamber through a plurality of liquid inlet branches, the volumes of the reaction chambers are the same, the reaction chambers are connected to the liquid outlet branches, the outlets of the liquid outlet branches are connected to the gas outlets, a water-proof and breathable pad is provided on the gas outlet, the gas outlet is sealed by a gas outlet pressure plate, and a light-transmitting window is provided below the reaction chamber.

7. The microbial nucleic acid detection microfluidic chip according to claim 6, characterized in that: The inlet of the reaction detection pool is connected to each of the liquid inlet branches through a liquid inlet main pipe. A bypass liquid flow channel is also connected in parallel on one side of the liquid inlet branch. The diameter of the bypass liquid flow channel is wider than the diameter of the liquid inlet branch. The outlet of the bypass liquid flow channel is connected to the air outlet, and the volumes of the liquid outlet branches are the same.

8. The microbial nucleic acid detection microfluidic chip according to claim 1, characterized in that: The heating component is provided with a heat conducting plate, a heating plate and a heat insulating plate from bottom to top. A temperature sensor is provided in the heat conducting plate and is sealed and fixed at a corresponding position on the microfluidic chip through a sealing ring.

9. The microbial nucleic acid detection microfluidic chip according to claim 1, characterized in that: An elliptical liquid level detection pool is arranged in the liquid inlet pipeline of the lysis pool, and metal electrodes are arranged at both ends of the liquid level detection pool, one end of the electrode is in the detection pool, and the other end leaks out of the chip and is connected to a wire.

10. The microbial nucleic acid detection microfluidic chip according to claim 9, characterized in that: The liquid inlet pipeline of the cracking tank is provided with a serpentine pipeline.

11. The microbial nucleic acid detection microfluidic chip according to claim 5, characterized in that: The connecting pipeline between the sample inlet and the degassing flow channel passes through the water-proof and air-permeable pad, and the pipeline passing through is sealed by the water-proof and air-permeable pad.

12. An automatic detection kit comprising the microbial nucleic acid detection microfluidic chip according to any one of claims 1 to 11, characterized in that: The automatic detection box is detachably connected to the top of the detection equipment box body by a chip box body, the microbial nucleic acid detection microfluidic chip is fixed inside the chip box body, the sampling chip is fixed inside the chip box body, the sample injection port abuts against the surface of the chip box body, and the sealing screw protrudes outside the chip box body; the fluorescence detector is arranged in the detection equipment box body, and the fluorescence probe of the fluorescence detector corresponds to the position of the reaction chamber. A main control board is also provided in the detection equipment box body, and the main control board is used to electrically control various electrical components.

13. The automatic detection box according to claim 12, characterized in that: The fluorescence detector is connected to the detection device box through a connecting rod. The connecting rod is provided with a rotating wheel and a belt. The rotating wheel is driven by a stepper motor to drive the belt to rotate, thereby completing the lateral movement of the fluorescence detector under each reaction chamber. The lateral movement range is limited by a photoelectric switch. The fluorescence detector is provided with FAM, VIC, ROX, and CY5 channels.

14. The automatic detection box according to claim 13, characterized in that: The main control board controls the heating of the heating plate and collects the temperature of the temperature sensor in the heat conducting plate, controls the opening and closing of the peristaltic pump and the solenoid valve, reads the liquid detection signal of the liquid level sensor, controls the rotation of the stepper motor and the fluorescence signal collection of the fluorescence detector, and reads the position signal of the photoelectric switch.

15. A method for detecting Staphylococcus aureus nucleic acid using the automatic detection kit according to claim 12, characterized in that: The following steps are involved: Step 1) pre-place LAMP amplification primers of the nuc gene of Staphylococcus aureus, sgRNA targeting the nuc gene of Staphylococcus aureus, AapCas12b protease, fluorescent probe, Bst3.0 polymerase, dNTPs reagent and lyophilized powder of reaction buffer in the reaction chamber; one of the reaction chambers is set as a blank control; Step 2) Open the sealing screw, and the swab carrying the sample to be tested enters the sample chamber from the sample loading port, break the swab rod and leave the swab head in the sample chamber, tighten the sealing screw, and the main control board controls the peristaltic pump to pump the eluent in the liquid storage bag into the eluent inlet, open the first solenoid valve, and the eluent flows into the sample chamber of the sample injection chip for elution, and then enters the degassing flow channel from the sample inlet to degas the eluent; Step 3) The main control board controls the peristaltic pump to continue pumping liquid. After the electrode detects that the eluent flows into the liquid level detection pool, the timing is started, and the peristaltic pump is controlled by time to transport the eluent to fill the lysis pool. Then the peristaltic pump, the first solenoid valve and the second solenoid valve are closed, and the heating component connected above the lysis pool is controlled to heat to 90°C, and the lysis is performed for 5-15 minutes to complete the nucleic acid extraction; Step 4) The main control board controls the peristaltic pump and the third solenoid valve to open, deliver the lysate into the reaction chamber, then closes the third solenoid valve, and controls the heating component connected above the reaction chamber to heat to 65° C., reacts for 60 minutes, and amplifies the nucleic acid at a constant temperature for 60 cycles, each cycle lasting 1 minute; Step 5) The main control board controls the stepper motor to drive the fluorescence detector to pass through the bottom of each reaction chamber in turn, and the fluorescence probe opens the FAM channel to perform fixed-point fluorescence detection. The main control board controls the fluorescence probe to perform a fluorescence signal detection once in each cycle.

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